The neurotransmitter dopamine activates two major classes of dopamine receptors. These include the D1-like (D1 and D5) and the D2-like dopamine receptors (D2, D3 and D4). Based on its expression pattern in the brain, and on studies using D3-preferring agonists, the D3 receptor is postulated to play a role in neurological disorders such as schizophrenia, Parkinson's disease, drug addiction, and depression. In heterologous expression systems, the D2-like dopamine receptors couple to and modulate adenylyl cyclases, ion channels and protein kinases. The in vivo coupling to signal transduction pathways has been more difficult to determine, particularly for the D3 dopamine receptor. The lack of agonists that can selectively distinguish the D3 dopamine receptor from other D2-like dopamine receptors, coupled with the limited expression profile of the D3 receptor has contributed to the inability to fully determine its in vivo signal transduction pathways and properties. Previous studies have been inconclusive or have shown different functional effects in different cell populations. The results to date suggest that in vivo D3 receptor function in the brain is not clear and likely exhibits region- and cell type-specific differences. In this R21 project we test the hypothesis that the D3 dopamine receptors in the brain couple to ion channels, exhibiting regional and cell type specific differences in signaling function that is determined by differential co expression with other dopamine receptor subtypes. We will test this hypothesis using the novel Drd3-EGFP transgenic mouse model generated by the NINDS Gene Expression Nervous System Atlas (GENSAT) Project. These transgenic mice express the fluorescent enhanced green fluorescent protein (EGFP) in cells natively expressing wild type D3 dopamine receptors, facilitating the identification and functional characterization of D3 receptors in vivo. Given its novelty, to date, this transgenic model has not been fully characterized;therefore in the first specific aim, we will determine the mRNA expression profile of D3 receptor and other dopamine receptor subtypes in pre- and post-synaptic cell types at the single cell level in five brain regions of the Drd3-EGFP transgenic mice. In the second specific aim we will compare the functional coupling of D3 receptors to ion channels at the single cell level in fluorescent cells isolated from the nucleus accumbens and substantia nigra brain regions of the Drd3- EGFP transgenic mice. These two regions have been previously reported to express D3 receptors at relatively high levels, and have also been implicated in neuropsychiatric and neurodegenerative disorders. This two year detailed R21 project will determine for the first time, the regional- and cell type- specific differences in D3 receptor signaling function in the brain. In addition, this project will validate the Drd3-EGFP transgenic mouse model for future studies of D3 receptor function in normal and disease states. Our long term goal is to use the Drd3-EGFP mice to study behavioral and molecular responses to pharmacological and genetic manipulation of D3 receptor and the proteins that constitutes its signaling pathways. PUBLIC HEALTH RELEVANCE: In this R21 project we will characterize the expression and function of the D3 dopamine receptors in vivo in a novel transgenic mice model. Characterization of D3 receptors in these transgenic mice will facilitate the future use of this model in behavioral and pharmacological studies that test novel drugs targeting the D3 receptor signal transduction pathways. Determination of in vivo function of D3 dopamine receptors will help understand its role in diseases such as schizophrenia, Parkinson's disease, drug addiction and depression.